International Biodeterioration & Biodegradation 90 (2014) 45e51

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Diversity of cyanobacteria on stone monuments and building facades

of India and their phylogenetic analysis
Nitin Keshari 1, Siba Prasad Adhikary*
Department of Biotechnology, Institute of Science, Visva-Bharati, Santiniketan 731235, West Bengal, India

a r t i c l e i n f o a b s t r a c t

Article history: Many archaeologically important stone temples, caves, mortar monuments with artistic expression as
Received 11 December 2013 well as building facades of India are now disfigured due to colonization of cyanobacterial biofilms leading
Received in revised form to weathering of the substratum. They are composed of species principally belonging to the genera
13 January 2014
Hassallia, Tolypothrix, Scytonema, Lyngbya and Calothrix, which appeared soon after wetting of the bio-
Accepted 22 January 2014
films. Several other species of genera Aulosira, Nostoc, Camptylonema, Dichothrix, Chlorogloeopsis and
Available online 6 March 2014
Westiellopsis occurred as associated organisms as they appeared upon prolonged culture of the biofilms.
Molecular phylogenetic analysis based on 16S rRNA partial gene sequencing of all these 24 cyanobacteria
Keywords:
Biofilms
species under 11 genera isolated from the surfaces of monuments and building facades of India along
Stone monuments with those of other species isolated from stone surfaces in subaerial habitats and hypogeal environments
Building facades in different regions of the globe showed that those species from tropical regions were clustered together,
Cyanobacteria quite different from cyanobacteria of the genera Chroococcidiopsis, Leptolyngbya, Phormidium, Nostoc,
Molecular phylogeny Rexia, Symphyonemopsis, Scytonema, Tolypothrix and Calothrix occurring in the temperate regions.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
The exposed surfaces of stone temples, monuments as well as
building facades in most regions of India look blackish colour due to
formation of phototrophic biofilms. The principal species there-in
are certain species of cyanobacteria (Roy et al., 1997; Tripathy
et al., 1997). They could survive in the biofilms even during sum-
mer months when the temperature on these structures exceeds
60
C coupled with high light intensity and extreme dryness
(Tripathy et al., 1999; Pattanaik and Adhikary, 2002; Samad and
Adhikary, 2008; Keshari and Adhikary, 2013). These organisms
sprout receiving moisture on the onset of monsoon and grow
utilizing the minerals leading to deterioration of monuments of
cultural value (Gaylarde and Morton, 1999; Gaylarde and Gaylarde,
2000, 2001; Crispim et al., 2003). Certain species of cyanobacteria
possess well developed sheath layers around their trichome con-
taining UV-sunscreen pigments like scytonemin and MAAs (Garcia-
Pichel and Castenholz, 1991, 1993; Adhikary and Sahu, 1998; Mandal
* Corresponding author. Tel.: þ91 3463261101.
E-mail addresses: nitindna@gmail.com (N. Keshari), adhikarysp@visva-bharati.
ac.in (S.P. Adhikary).
1
Tel.: þ91 3463261101.
and Rath, 2012) and secrete copious extracellular polymeric sub-
stances (EPS) (Bertocchi et al., 1990; Gloaguen et al., 1995; Adhikary,
1998; Rossi et al., 2012), all of which contribute to their protection
against desiccation and intense solar radiation. Due to their photo-
trophic nature and many being nitrogen fixers, cyanobacteria
colonize easily on exposed surfaces that in due course lead to for-
mation of patinas and incrustations causing aesthetic damage.
Cyanobacteria being important from evolutionary and ecological
point of view, the mode of their diversity analysis and taxonomy are
changing with recent information and techniques. Very little in-
formation is available on identification and phylogeny of cyano-
bacteria species from Indian environments following molecular
approach (Sahu and Adhikary, 2012; Keshari and Adhikary, 2013).
Organisms occurring in desiccated state on sub-aerial surfaces are
difficult to identify following available monographs as seldom the
morphological features of the species can be seen even after prolong
wetting of the natural material. Further, when adapted to extreme
environments they undergo structural adaptation, thus identifica-
tion and determining their evolutionary relationship is difficult.
Hence 16S rRNA gene sequences of cyanobacteria species isolated
from stone temples and monuments as well as on building facades
from several locations of India were obtained and their molecular
phylogeny was determined comparing the gene sequences of spe-
cies from identical surfaces of other regions of the globe.

http://dx.doi.org/10.1016/j.ibiod.2014.01.014
0964-8305/Ó 2014 Elsevier Ltd. All rights reserved.
46 N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e51

Table 1
List of major and associated species of cyanobacteria isolated from the exterior of different stone monuments, caves and building facades of India.

Cyanobacteria Place of collection Substratum Major Associated

species species

Hassallia byssoidea Hassall (Fig. 2, a) Sun temple, Konark, Odisha Stone surface þ
Dichothrix baueriana Bornet et Flahault (Fig. 2, b) Sun temple, Konark, Odisha Stone surface þ
Tolypothrix scytonemoides (Gardner) Geitler (Fig. 2, c) Kedar-Gouri temple, Bhubaneswar, Odisha Stone surface þ
Nostoc carneum Agardh ex Bornet et Flahault (Fig. 2, d) Kedar-Gouri temple, Bhubaneswar, Odisha Stone surface þ
Tolypothrix distorta var. penicillata Kützing ex Bornet Jogin temple, Hirapur, Bhubaneswar, Odisha Stone surface þ
et Flahault (Fig. 2, e)
Nostoc commune Voucher ex Bornet et Flahault (Fig. 2, f) Lingaraj temple, Bhubaneswar, Odisha Stone surface þ
Nostoc linckia (Roth) Bornet ex Bornet et Flahault (Fig. 2, g) Rameswar temple, Bhubaneswar, Odisha Stone surface þ
Lyngbya aerugineo-coerulea (Kützing) Gomont (Fig. 2, h) Brahmeswar temple, Bhubaneswar, Odisha Stone surface þ
Nostoc punctiforme (Kützing) Hariot (Fig. 2, i) Nrusinghanath temple, Paikamal, Odisha Stone surface þ
Chlorogloeopsis fritschii Mitra et Pandey (Fig. 2, j) Vrihadeswar temple, Thanjavur, Tamil Nadu Stone surface þ
Lyngbya kuetzingiana Kirchner (Fig. 2, k) Tiger cave, Khandagiri, Bhubaneswar, Odisha Stone surface þ
Tolypothrix bouteillei (Brebissom et Desm.) Forti (Fig. 2, l) Ajanta caves, Maharashtra Stone surface þ
Calothrix gardneri De Toni (Fig. 2, m) Ajanta caves, Maharashtra Stone surface þ
Scytonema coactile Montagne ex Bornet et Flahault (Fig. 2, n) Kutumsar cave, Chhattisgarh Stone surface þ
Scytonema millei Bornet ex Bornet et Flahault (Fig. 2, o) Buddha statue, Santiniketan, West Bengal Stone and mortar þ
Scytonema chiastum Geitler (Fig. 2, p) Buddha statue, Santiniketan, West Bengal Stone and mortar þ
Westiellopsis prolifica Janet (Fig. 2, q) Buddha and Gandhi statue, Santiniketan, Stone and mortar þ
West Bengal
Tolypothrix campylonemoides Ghose (Fig. 2, r) Gandhi statue, Santiniketan, West Bengal Stone and mortar þ
Aulosira pseudoramosa Bharadwaja (Fig. 2, s) Gandhi statue, Santiniketan, West Bengal Stone and mortar þ
Scytonema sp. (Fig. 2, t) Elephant sculpture, Santiniketan, West Bengal Stone and mortar þ
Calothrix marchica lemmermann (Fig. 2, u) Rabindra Bhavana and Siksha Bhavana, Building Facades þ
Santiniketan, West Bengal
Scytonema rivulare Borzi ex Bornet et Flahault (Fig. 2, v) Patha Bhavana, P.M. hospital, Silpa Sadan Building Facades þ
and Santisree hostel, Santiniketan, West Bengal
Camptylonema indicum Schmidle (Fig. 2, w) Siksha Bhavana, Santiniketan, West Bengal Building Facades þ
Tolypothrix rechingeri (Wille) Geitler (Fig. 2, x) Siksha Bhavana, Santiniketan, West Bengal Building Facades þ

2. Material and methods

2.1. Description of sampling sites

Eight stone temples, three caves, three mortar monuments and

six building facades from different regions of India (Table 1, Fig. 1)
were sampled for biofilms occurring on their exterior surfaces. Of
these Sun temple of Konark; Kedar-Gouri, Jogin, Lingaraj, Rames-
war and Brahmeswar temples of Bhubaneswar (19
800 to 20
240 N,
85
870 to 86
90 E) and Nrusinghanath temple of Paikamal (20
680 N,
82
460 E) were from Odisha and Vrihadeswar temple of Thanjavur
(10
460 N, 79
70 E) from Tamil Nadu. These were built during 6th to
16th A.D., hence important from archaeological point of view. Be-
sides the Tiger cave of Khandagiri (20
200 N, 85
440 E) from Odisha,
Ajanta caves (20
330 N, 75
420 E) famous for artistic expressions and
fresco-paintings from Maharashtra, Kutumsar cave (19
070 N,
82
030 E) from Chhattisgarh and the monuments made of mortar
and the building facades of Santiniketan (23
680 N, 87
680 E), a
cultural heritage site in West Bengal were also sampled.

2.2. Collection, observation, culturing and identification

Blackish-brown biofilms were collected from all the sampling

locations soon after monsoon rain and stored in pre-sterilized
sampling bottles. During the other seasons it was difficult to
remove the biofilms from the stone surfaces, hence were collected
following non-destructive method by employing adhesive tape
strips (La Cono and Urzi, 2003). The biofilms were soaked in sterile
distilled water and incubated under fluorescent light for up to 72 h
and observed microscopically. Since the morphological features
needed for identification were not distinct even after prolonged
Fig. 1. Map of India showing sampling sites for cyanobacterial biofilms on exterior
soaking (up to 7 days), a small amount of each sample was trans- surfaces of temples and monuments in different locations of India. 1: Santiniketan (West
ferred to BG 11 medium with and without nitrogen (Rippka et al., Bengal), 2: Paikamal (Odisha), 3: Bhubaneswar (Odisha), 4: Konark (Odisha), 5: Kutumsar
1979) and to agar plates (1.2% w/v agar in the same medium). cave (Chhattisgarh), 6: Ajanta caves (Maharashtra) and 7: Thanjavur (Tamil Nadu).
 N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e51 47

Fig. 2. Microphotographs of cyanobacteria isolated from the exterior of different stone monuments, caves and building facades of India. a. Hassallia byssoidea, b. Dichothrix
baueriana, c. Tolypothrix scytonemoides, d. Nostoc carneum, e. Tolypothrix distorta var. penicillata, f. Nostoc commune, g. Nostoc linckia, h. Lyngbya aerugineo-coerulea, i. Nostoc
punctiforme, j. Chlorogloeopsis fritschii, k. Lyngbya kuetzingiana, l. Tolypothrix bouteillei, m. Calothrix gardneri, n. Scytonema coactile, o. Scytonema millei, p. Scytonema chiastum, q.
Westiellopsis prolifica, r. Tolypothrix campylonemoides, s. Aulosira pseudoramosa, t. Scytonema sp., u. Calothrix marchica, v. Scytonema rivulare, w. Camptylonema indicum, x. Tolypothrix
rechingeri. Scale bar: Fig. aex ¼ 10 mm.
48 N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e51

Cultures were incubated at 25  1
C under continuous light from
fluorescent tubes at an intensity of 7.5 W m2. The cyanobacteria
species appeared in the culture were isolated and maintained in the
laboratory. Microphotographs were taken using an Olympus BX 41
microscope fitted with a Nikon Coolpix 4500 digital camera. These
were identified following standard monographs (Geitler, 1925;
Desikachary, 1959; Hindák, 2008) and related articles (Komárek
and Anagnostidis, 1989; Anagnostidis and Komárek, 1990; Dodds
et al., 1995).
2.3. Genomic DNA extraction, amplification of 16S rRNA gene and
phylogenetic analysis
Genomic DNA was extracted from 24 different cyanobacteria
species isolated into culture (Fig. 2) following a bacterial genomic
DNA isolation protocol. Exponentially growing cells were pelleted
by centrifugation and resuspended in 567 ml lysis buffer (10 mM
TriseHCl, pH 8.0, 1 mM trisodium citrate and 1.5% SDS) followed by
incubation for 1 h at 37
C after addition of 30 ml of 10% SDS and 3 ml
of 20 mg ml1 proteinase K. To this, 100 ml 5 M NaCl was added,
followed by addition of 80 ml CTAB/NaCl solution (10% CTAB/0.7 M
NaCl) and incubated for 10 min at 65
C. The lysate was extracted
with equal volume of chloroform:isoamyl alcohol (24:1) and then
an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1).
DNA was precipitated with 0.6 volume isopropyl alcohol, washed
with 70% ethanol, air dried and resuspended in 100 ml TE buffer.
PCR amplification of 16S rRNA genes of the experimental organisms
was carried out using CYA359F and CYA781R (equimolar mixture of
CYA781R-a and CYA781R-b) primers specific for cyanobacteria
(Nübel et al., 1997). Template DNA (10 ng) was added to the reac-
tion mixture in a total volume of 50 ml containing 31 ml milli Q
water, 5 ml 10 Buffer (15 mM MgCl2), 2 ml dNTPs (10 mM), 2.5 ml
forward primer CYA359F and 1.25 ml each of reverse primer
CYA781R-a and CYA781R-b, 1 ml Taq polymerase and 1 ml BSA
(20 mg ml1). Amplification was performed using the PCR system
9700 (Applied Biosystems). The products were purified using a
Qiagen gel extraction kit and sequenced (GCC Biotech Company,
Kolkata). The sequences of 16S rRNA of all the species were

Table 2
Cyanobacteria isolated from the exterior of monuments of different regions of India and their accession number after deposition in NCBI GenBank and details of accession
number of other cyanobacteria from stone surfaces in subaerial habitats and hypogeal environments in different regions of the globe retrieved from GenBank used for
construction of Consensus tree.

Cyanobacteria Accession no Location References

Leptolyngbya sp. AY769961 Catacombs, Italy Bruno et al., 2009

Leptolyngbya sp. DQ295209 Catacombs, Italy Bruno et al., 2009
Leptolyngbya sp. DQ295210 Catacombs, Italy Bruno et al., 2009
Leptolyngbya sp. DQ295207 Catacombs, Italy Bruno et al., 2009
Leptolyngbya sp. DQ295208 Catacombs, Italy Bruno et al., 2009
Chroococcidiopsis sp. AF279110 Beacon sandstone, Antartica Billi et al., 2001
Chroococcidiopsis sp. AF279107 Rock crust in Nubian sandstone, Israel Billi et al., 2001
Chroococcidiopsis sp. AF279108 Granite, Egypt Billi et al., 2001
Chroococcidiopsis sp. AF279109 Granite boulder, Chile Billi et al., 2001
Rexia erecta AY452533 Great Smoky Mountains National Park, USA Casamatta et al., 2006
Phormidium sp. AM398795 Rock surface, Switzerland Marquardt and Palinska, 2007
Phormidium sp. AM398775 Rock surface, Italy Marquardt and Palinska, 2007
Leptolyngbya sp. AM398803 Stone (Granite), Nepal Marquardt and Palinska, 2007
Phormidium tergestinum AM398776 Mortar, Spain Marquardt and Palinska, 2007
Nostoc cf. commune HQ877825 Building walls, Mexico Ramírez et al., 2011
Symphyonemopsis sp. AJ544085 Cave, Spain Gugger and Hoffmann, 2004
Scytonema sp. DQ531701 Biological soil crust, Cyanolands National Park, USA Yeager et al., 2007
Tolypothrix sp. HM751852 Rock surface of siliceous stream, Madrid, Spain Berrendero et al., 2011
Calothrix sp. HM751844 Rock surface of siliceous stream, Madrid, Spain Berrendero et al., 2011
Calothrix sp. HM751845 Rock surface of siliceous stream, Madrid, Spain Berrendero et al., 2011
Gloeobacter violaceus AF132790 PCC 7421 Turner et al., 1999
Fischerella muscicola KC736951 Stone temple, Nepal Unpublished
Hassallia byssoidea KC736947 Stone temple, Odisha, India Present work
Dichothrix baueriana KC736961 Stone temple, Odisha, India Present work
Tolypothrix scytonemoides KC736948 Stone temple, Odisha, India Present work
Nostoc carneum KC736962 Stone temple, Odisha, India Present work
Tolypothrix distorta var. penicillata KC736949 Stone temple, Odisha, India Present work
Nostoc commune KC736958 Stone temple, Odisha, India Present work
Nostoc linckia KC736960 Stone temple, Odisha, India Present work
Lyngbya aerugineo-coerulea KC736956 Stone temple, Odisha, India Present work
Nostoc punctiforme KC736959 Stone temple, Odisha, India Present work
Chlorogloeopsis fritschii KC736950 Stone temple, Tamil Nadu, India Present work
Lyngbya kuetzingiana KC924443 Tiger cave, Odisha, India Present work
Tolypothrix bouteillei KC736953 Ajanta caves, Maharashtra, India Present work
Calothrix gardneri KC736954 Ajanta caves, Maharashtra, India Present work
Scytonema coactile KC736955 Kutumsar cave, Chhattisgarh, India Present work
Scytonema millei JX523941 Stone and mortar monument, West Bengal, India Present work
Scytonema chiastum JX523943 Stone and mortar monument, West Bengal, India Present work
Westiellopsis prolifica JX523946 Stone and mortar monument, West Bengal, India Present work
Tolypothrix campylonemoides JX523942 Stone and mortar monument, West Bengal, India Present work
Aulosira pseudoramosa JX523944 Stone and mortar monument, West Bengal, India Present work
Scytonema sp. JX523945 Stone and mortar monument, West Bengal, India Present work
Calothrix marchica KC924438 Building facades, West Bengal, India Present work
Scytonema rivulare KC924440 Building facades, West Bengal, India Present work
Camptylonema indicum KC924439 Building facades, West Bengal, India Present work
Tolypothrix rechingeri KC924441 Building facades, West Bengal, India Present work
 N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e51 49

deposited in the GenBank database with accession no. JX523941
to JX523946, KC736947 to KC736950, KC736953 to KC736956,
KC736958 to KC736962, KC924438 to KC924441 and KC924443.
The 16S rRNA sequences of other cyanobacterial species from sub-
aerial habitats reported earlier were retrieved from GenBank for
construction of consensus tree following maximum parsimony
analysis using Mega-4.0 software (Tamura et al., 2007). Gloeobacter
violaceus PCC 7421 was used as the outgroup (Turner et al., 1999).
Number near node represents bootstrap value was obtained from
1000 replicates.

Fig. 3. Consensus tree from Maximum Parsimony showing the phylogenetic relationship between the cyanobacteria isolated from biofilms on exterior surfaces in different locations
of India and the organisms from other regions of the globe based on the 16S rRNA partial gene sequences. Organisms from India were depicted with different symbols depending
upon the types of monuments from where those were isolated (C: stone and mortar monuments, A: stone temples, -: caves, :: building facades).
50 N. Keshari, S.P. Adhikary / International Biodeterioration & Biodegradation 90 (2014) 45e51
3. Results and discussion
The blackish brown biofilms on the exterior of monuments in
different location of India were comprised of 24 species under 11
genera (Table 1, Fig. 2). Hassallia byssoidea was the major cyano-
bacterium on Sun temple, Konark, whereas species belonging to
Tolypothrix, Scytonema and Lyngbya were the major organisms on
several temples of Bhubaneswar, caves of Maharashtra, Chhattis-
garh and Odisha and also on the mortar monuments of Santinike-
tan in West Bengal. Other cyanobacteria under the same genera and
in addition, species of Nostoc, Calothrix and Westiellopsis appeared
along with the major cyanobacteria upon prolonged culture, hence
were designated as associated organisms (Table 1). A species of
Calothrix, Calothrix marchica was the major organism on building
facades of Santiniketan and one species each of Tolypothrix and
Camptylonema occurred as the associated components. In general
filamentous cyanobacteria with thick sheath layer around their
trichomes were the major species occurring on the exposed stone
and mortar surfaces forming biofilms in Indian environments
(Tripathy et al., 1997, 1999; Pattanaik and Adhikary, 2002).
For molecular phylogeny analysis 16S rRNA partial gene se-
quences of all these 24 species of cyanobacteria obtained in culture
were generated and deposited in GenBank (Table 2). The 16S rRNA
sequences of cyanobacteria species from sub-aerial habitats re-
ported so far from across the globe were retrieved from GenBank
and used to make a consensus tree. These organisms were isolated
from Catacombs of Italy, stone/rock surfaces of Israel, Egypt, Chile,
USA, Switzerland, Nepal, Spain, and Antarctica, caves of Spain,
building facades of Mexico, biological soil crusts of USA as well as
rock surface of siliceous stream of Spain covering the temperate as
well as tropical zones (Billi et al., 2001; Gugger and Hoffmann,
2004; Casamatta et al., 2006; Marquardt and Palinska, 2007;
Yeager et al., 2007; Bruno et al., 2009; Berrendero et al., 2011;
Ramírez et al., 2011). Maximum parsimony analysis showing
phylogenetic relationships between all these cyanobacteria species
is given in Fig. 3. Sequence accession numbers of the organisms
obtained from GenBank are given in parenthesis. Analysis of the
results showed that cyanobacteria under Nostocales and Stigone-
matales from different localities of India formed a genera-wise
monophyletic clade i.e. Lyngbya kuetzingiana and Lyngbya aerugi-
neo-coerulea (Fig. 3, clad-VII); Calothrix gardneri, Calothrix marchica
and Dichothrix baueriana (Fig. 3, clad-I); and Westiellopsis prolifica,
Fischerella muscicola with Chlorogloeopsis fritschii (Fig. 3, clad-V).
Likewise Hassallia byssoidea and species of Nostoc formed a sepa-
rate clade (Fig. 3, clad-VI), species of Tolypothrix, Scytonema,
Camptylonema almost clustered together (Fig. 3, clad-II, III, IV) while
Aulosira pseudoramosa clustered with other heterocystous cyano-
bacteria. Thus, phylogenetic clustering supported morphological
characteristics in most cases. Further, the resultant tree showed
that the cyanobacteria in the biofilms on the exterior of stone
monuments in tropical climatic regime like India were clustered
quite differently from the species under genera Chroococcidiopsis
(Fig. 3, clad-IX), Leptolyngbya (Fig. 3, clad-VIII), Nostoc, Rexia, Sym-
phyonemopsis, Tolypothrix, Scytonema and Calothrix (Fig. 3, clad-X)
with Phormidium in-between isolated from stone surfaces under
different climatic regime.
4. Conclusions
These results showed that different species of cyanobacteria
form blackish brown biofilms on stone surfaces of monuments as
well as building facades globally (Pattanaik and Adhikary, 2002;
Crispim et al., 2004; Ortega-Morales et al., 2005; Pereira de Oli-
veira et al., 2011) and are responsible for their biodeterioration
(Ortega-Calvo et al., 1991; Gaylarde and Morton, 1999; Gaylarde and
Gaylarde, 2000, 2001; Ramírez et al., 2010). The species that occur
on such surfaces were not similar phylogenetically. Morphological
as well as molecular techniques are essential to understand the
colonization pattern of cyanobacteria on exterior of stone monu-
ments and building facades exposed to extreme heat and desicca-
tion, and their role in biodeterioration.
Acknowledgements
We are grateful to the Department of Science and Technology
(DST), Government of India for financial assistance through a
Cluster project on “Genomics and Proteomics of stress tolerant
cyanobacteria”. We thank the authorities of Visva-Bharati, Santi-
niketan for providing laboratory facilities.
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